title: Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing.
abstract: OBJECTIVE
  To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect.
  
  
  METHODS
  The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated.
  
  
  RESULTS
  Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM.
  
  
  CONCLUSIONS
  These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okumura
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Nakano
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
authors.forename: Ryohei
authors.initials: R
authors.lastname: Numata
authors.forename: Aya
authors.initials: A
authors.lastname: Ota
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sowa
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sakai
authors.forename: Morio
authors.initials: M
authors.lastname: Ueno
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D056741:Cyclin D / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005131:Eye Injuries / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D008252:Macaca fascicularis; D011725:Pyridines / Q000494:pharmacology*; D011817:Rabbits; D015398:Signal Transduction; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D000577:Amides; D056741:Cyclin D; D004791:Enzyme Inhibitors; D011725:Pyridines; C520365:Y-39983; C108830:Y 27632; D050760:Cyclin-Dependent Kinase Inhibitor p27
keywords: ROCK inhibitor; bullous keratopathy; cell proliferation; corneal endothelial cells